In-vitro detection of reactions in blood to foreign substances

ABSTRACT

The present invention is a method of detecting reaction in blood caused by the presence of a foreign substance in the blood, comprising the steps of: establishing a potential across a predetermined spatial volume; passing a first portion of the blood through the predetermined spatial volume; substantially continuously measuring the potential across the predetermined spatial volume over a first predetermined period of time; comparing the measured potential with a baseline; and calculating the total volume of solids in the first portion of the blood as a function of a total absolute deviation of the measured potential from the baseline. The same procedure is then followed with a second portion of the blood, after it has been exposed to the substance whose reaction is being determined. The two calculations are then compared, with a positive reaction being indicated when the two measured solid volumes are measurably different. The baselines are preferably dynamic baselines, and are determined with reference to the starting point of a sharp rise in the measured potential.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of my prior filed PCTapplication, Ser. No. PCT/US96/12629, filed Aug 1, 1996 (the disclosureof which is hereby incorporated by reference), which was itself acontinuation-in-part of a prior filed provisional application, Ser. No.60/001,824, filed Aug 1, 1995.

This application is also a continuation-in-part of my prior filed U.S.provisional application Ser. No. 60/014,060 filed Mar. 25, 1996.

Priority of these applications is claimed herein.

BACKGROUND OF THE INVENTION

The present invention is directed to the field of medical diagnoses,and, more specifically, diagnoses performed by detecting reactions inblood caused by the presence of foreign substances therein.

I refer to this test as the "MRT" Test.

The MRT Test relates to the field of hypersensitivity reactions observedin humans and animals. These reactions can be due to contact withoffending substances such as medications, environmental chemicals,foods, carcinogens, food additives, etc.

The MRT Test is an in-vitro assay which indirectly detects the releaseof mediators in whole blood after it is mixed with a test substance.When a patient's blood reacts with the test substance, intracellularfluids are released, causing the liquid portion of blood to increase,while the total volume of the solids present in the blood decreases.These reactions may be caused by various immunologic and non-immunologicmechanisms.

Accordingly, it is an objective of this invention to provide an in-vitromethod which will identify reactions caused by various test substances.

It is also an objective of this invention to identify the volumetricdifferences in the level of plasma in non-treated blood vs. the level ofplasma in treated blood.

It is a further objective of this invention to use this new laboratorymethod as a unique way to solve the problem of identifying maladieswhich are otherwise difficult to diagnose.

About blood:

Blood is a liquid that circulates throughout the body using the vascularsystem and is in contact with practically every cell in the body. Blooddelivers oxygen, food and other essential elements to all of our cells.Approximately 50% of blood is a fluid called serum (or plasma). It is acomplex mixture of water, various proteins, carbohydrates, lipids, andelectrolytes. Small amounts of other substances such as vitamins.Minerals, and hormones are also found in blood. The other 50% of bloodis comprised of solids such as erythrocytes (red blood cells: RBC),leukocytes (white blood cells: WBC), and thrombocytes (Platelets).

The white blood cells are a significant part of our body's immunesystem. The immune system is highly complex and intricate in its designand is responsible for defending against foreign invaders such asbacteria, viruses, and other pathogens. The science of immunologyincorporates the study of resistance to infections and the rejections ofso called "foreign substances".

Gell and Coombs in their 1962 book, Clinical Aspects of Immunology, haveidentified various immune mediated hypersensitivity reactions andcategorized them as Types I-IV, based upon the mechanics of thereaction. Types I, II. and III are identified as antibody mediated andthe fourth one is described as cell mediated.

It is understood that Type I is the most widely occurringhypersensitivity reaction. It involves Mast cells and basophils, whichbind IgE through their Fc receptors. After encountering the antigen, theantibody induces degranulation (the destruction of the exterior wall ofthe cell) and release of mediators.

Type II reactions involve the binding of antigen and antibody on thesurface of a cell, generally resulting in the destruction of the cell.As is the case in a Type I reaction, the final outcome of this reactiongenerates the release of cellular contents (including the release of themediators).

Type III reactions address the interactions of cells with complexes.Immune complexes, when deposited on tissue, cause complement activation,which in turn attracts polymorphonuclear leukocytes ("polymorphs"). Astheir normal response, the polymorphs will attempt phagocytosis on thecomplexes, but in many instances the complexes will be trapped by thetissue, blocking phagocytosis. As a natural course, polymorphs willrelease inflammatory mediators.

Type IV reactions involve sensitized T-lymphocytes. After the secondcontact with a specific antigen. T cells release lymphokines, whichproduces an inflammatory response, and in turn attractsmediator-releasing macrophages.

This is an accepted theory, which generally explains the partial releaseof cytoplasm and mediators into the blood stream, or upon tissue as aresult of such reactions. As these reactions occur, the volumetric levelof the plasma will change.

As observed under the microscope, there are two possible reactionstriggered by offending substances;

a. release of liquid (substance, cytoplasm and mediators) from cells,causing decrease in solids/liquid volumetric ratio in blood; or

b. consumption of liquids, causing increase of solids/liquid ratio inblood.

It appears that at any time human blood can react one way or the otherHowever, it was also observed that usually only one type of reactiontakes place at a time.

It is contemplated that similar phenomena takes place by reason ofcontact with chemical substances such as gases (aerosols, pesticides,gases, cigarette fumes), paints, perfumes, oils, gas, thinners, airfresheners, food additives, drugs, and many other substances.

There is very little scientific explanation why humans and animals reactto the above named substances. Some theories suggest a classic allergicreaction, while others state that lack of specific enzymes, helping toneutralize foreign substances, are the reason for those reactions.

In summary, reactions caused by immune, toxic, pharmacological and othermechanisms may cause the release of mediators into the blood stream.

Current methods of diagnosis exist for measuring the degree of reactiona patient's blood may have with a suspected allergen, by measuring thesize and number of blood cells in a patient's blood. Such tests aredescribed in my prior U.S. Pat. Nos. 4,614,722; 4,788,155; and5,147,785, the disclosures of which are herein incorporated byreference. In essence, these patented tests operate by comparing thenumber and size of cells in a patient's blood before and after exposureto a foreign substance. If there is a significant cellular shift afterexposure, then a positive reaction is determined.

These tests, while a significant improvement in the art at the time theywere made, have a drawback, in that they do not well measure smalldifferences in cell sizes caused by the described cellular reactions.

Currently, no tests are known which may test for the reaction bloodcells have to foreign substances resulting in changes in plasma volumeindependent of changes in cell size distributions.

OBJECTS AND SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide an improvedmethod of determining cellular reaction caused by foreign substances,which overcomes the drawbacks of the prior art.

It is a further object of the invention to provide an improved methodfor diagnosing maladies caused by the presence of foreign substances ina patient's blood, by measuring the volume of plasma, or the volume ofsolids, in a patient's blood before and after exposure to a foreignsubstance whose effects are under consideration.

Briefly stated, there is provided a method of detecting reaction inblood caused by the presence of a foreign substance in the blood,comprising the steps of: establishing a potential across a predeterminedspatial volume; passing a first portion of the blood through thepredetermined spatial volume; substantially continuously measuring thepotential across the predetermined spatial volume over a firstpredetermined period of time; comparing the measured potential with abaseline; and calculating the total volume of solids in the firstportion of the blood as a function of a total absolute deviation of themeasured potential from the baseline. The same procedure is thenfollowed with a second portion of the blood, after it has been exposedto the substance whose reaction is being determined. The twocalculations are then compared, with a positive reaction being indicatedwhen the two measured solid volumes are measurably different. Thebaselines are preferably dynamic baselines, and are determined withreference to the starting point of a sharp rise in the measuredpotential.

In accordance with these and other objects of the invention, there isprovided an in-vitro method for detecting a reaction in blood caused bysubstances, comprising the steps of: establishing a first potentialacross a first predetermined spatial volume; passing a first portion ofthe blood through the first predetermined spatial volume; substantiallycontinuously measuring the first potential over a first predeterminedperiod of time; comparing the measured first potential with a firstbaseline; calculating the total volume of solids in the first portion ofthe blood as a first function of a total absolute deviation of themeasured first potential from said first baseline; exposing a secondportion of the blood to a substance; establishing a second potentialacross a second predetermined spatial volume; passing the second portionof the blood through the second predetermined spatial volume;substantially continuously measuring the second potential over a secondpredetermined period of time; comparing the measured second potentialwith a second baseline; calculating the total volume of solids in thesecond portion of the blood as a second function of a total absolutedeviation of the measured second potential from the second baseline; andcomparing the total volume of solids in the second portion of the bloodwith the total volume of solids in said first portion of blood, wherebya positive reaction is established when the total volume of solids inthe second portion of blood differs from the total volume of solids inthe first portion of blood by more than a predetermined error factor.

According to feature of the invention, there is further provided anin-vitro method for detecting a reaction in blood caused by substances,comprising the steps of: establishing a first potential across a firstpredetermined spatial volume; passing a first portion of the bloodthrough the first predetermined spatial volume; substantiallycontinuously measuring the first potential over a first predeterminedperiod of time; comparing the measured first potential with a firstdynamic baseline; calculating the total volume of solids in the firstportion of the blood as a first function of a total absolute deviationof the measured first potential from the first dynamic baseline;exposing a second portion of the blood to a substance; establishing asecond potential across a second predetermined spatial volume; passingthe second portion of the blood through the second predetermined spatialvolume; substantially continuously measuring the second potential over asecond predetermined period of time; comparing the measured secondpotential with a second dynamic baseline; calculating the total volumeof solids in the second portion of the blood as a second function of atotal absolute deviation of the measured second potential from thesecond dynamic baseline; and comparing the total volume of solids in thesecond portion of the blood with said total volume of solids in thefirst portion of blood, whereby a positive reaction is established whenthe total volume of solids in the second portion of blood differs fromthe total volume of solids in the first portion of blood by more than apredetermined error factor.

The above, and other objects, features and advantages of the presentinvention will become apparent from the following description read inconjunction with the accompanying drawings, in which like referencenumerals designate the same elements.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates an idealized particle (balloon) having a volume of300 μl, in a unit volume of 1 ml of a suspension, leaving a liquidvolume of 700 μl.

FIG. 2 illustrates an identical unit volume of 1 ml (not drawn toscale), in which the particle has a volume of only 100 μl, and theliquid a resultant volume of 900 μl.

FIG. 3 illustrates an actual oscilloscope reading of a series ofparticles being measured as they pass through the electromagnetic fieldunder observation.

FIG. 4 shows a close up of some oscilloscope readings such as depictedin FIG. 3.

FIG. 5 shows a smoothed curve showing three particles passing throughthe electromagnetic field being measured.

FIG. 6 shows an idealized representation of a series of particlespassing through the electromagnetic field.

FIG. 7 shows an idealized representation of a comparison of test andcontrol sample readings as the particles pass through theelectromagnetic field.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The MRT Test relies in large part upon the performance of the testdescribed in my co-pending PCT application, and reference is madethereto for a more complete understanding of the mechanics of thetesting being done. The following is presented for convenience ofreference.

Description of the MRT procedure:

1. Supplies and Instrumentation.

2. Blood collection and test preparation.

3. Testing.

4. Results.

1. Supplies and Instrumentation.

(supplies and instrumentation may vary to some extent and depend on thetype of testing instrument employed for the MRT Test. In this case Ihave chosen the semi-automated STS100 manufactured by Signet DiagnosticCorporation, and the following description is made with that device as areference).

100 μl-500 μl adjustable multi pipette

10-20 ml dispenser, e.g. an Oxford pipetor to dispense the electrolyticsolution mixed with a lysing agent

body temperature incubator, e.g. by Precision Scientific

60-100 rpm rotator, e.g. by Roto Mix

70 ml blood dilution vial with diluent

10 lysing reagent (as described in my prior patents) 8 ml vial

testing cuvettes with reagents. The reagents are dried and diluted foodextracts, e.g. by ALK or Bayer. Their concentration varies from 1:400 to1:2,000.000 depending upon their toxicity

isotonic (electrolytic) solution, e.g. Osmocel Isotonic Solution byHematronic

Apparatus, STS100 or STS200 made by Signet Diagnostic Corp.

2. Blood collection and test preparation.

I. Draw 5-10 ml of blood into a vacutainer containing 3.8% citratesolution without the "buffer" (citric acid, which may, itself, be anallergen).

II. Pour collected blood into the blood dilution vial.

III. Using the multi-pipette, transfer 700 μl of diluted blood intopanels of control and sample cuvettes (each panel will have at least onecontrol cuvette and at least one sample cuvette). Control samplescontain no reagent. Test samples contain a small amount of a substancebeing evaluated, the "reagent". The control sample serves as afingerprint of the patient's blood. The test sample provides informationrelated to the reaction of the tested substance to the reagent beingtested.

IV. After transferring blood to all tested samples, mix all cuvettes andcap them.

V. Place tray on the top of the rotator in the incubator. Turn therotator on.

VI. Incubate for 30 minutes at body temperature.

VII. Remove from incubator and follow by 30 minutes room temperatureincubation. Total of 60 minutes incubation.

3. Testing:

The MRT Test, the new proprietary laboratory method, can be described inthe following fashion:

a. Incubation of predetermined amount of blood in its native form whichserves as the fingerprint for the test.

b. Incubation of a predetermined amount of blood mixed with testedsubstance (at least 1 test sample).

c. Measure total volume of liquid and/or solids in native blood sampleby means of the method described in my prior PCT application.

d. Measure total volume of liquid and/or solids in the mixture of bloodand tested substance sample. If in step "c", you measure liquids, thendo so here. Likewise with solids, so that comparisons may be made"like-to-like".

e. Repeat step "d" for each tested substance. This may be done inparallel, i.e. several test measurements taken at the same time, or oneafter another. The parallel arrangement, however, is the mosttime-effective.

f. Identify volumetric differences of liquid volume and or solid volumebetween native blood sample and the tested blood sample.

g. Prepare the results identifying the measured volumetric differences.

h. Identify the positive and negative reactions, by noting whichreagents produced a measurable reaction, i.e. one greater than thestandard deviation expected for the test, calculated in known fashion.

The in vitro trend in the field of allergy, is to measure levels ofspecific immunoglobulins and detect the presence of individualmediators. In my research studies, I have identified that more then onemechanism may be employed in adverse reactions to foreign substances. Bymeasurement of volumetric differences in plasma we may deliver morecomprehensive answers.

FIG. 1 represents a small cuvette containing 1 ml of heterogeneousfluid. The liquid portion is equivalent to 700 μl. The balloon filledwith black ink has an equivalent volume of 300 μl. Note that forpurposes of measurement the balloon would be considered as a solidentity. FIG. 2 represents the same cuvette after the balloon hasreleased 200 μl of its ink into the external liquid. The Total volume ofsuspension is still 1 ml. The volume of the liquid has increased to 900μl and the volume of the balloon has decreased to 100 μl.

This example illustrates how human blood cells react in the body. Whenthe reacting substance is introduced to the blood, it triggers a seriesof complex reactions. In the end, the intracellular fluids will bereleased into the plasma, changing the original ratio of solids toliquid. The ratio is the key for identifying the malady (theintracellular liquids contain the mediators causing the clinicalsymptomology), but the ratio can be determined easily from a measurementof either the solid or the liquid volume per unit volume of the bloodsuspension.

There are many instruments widely used in the field of hematology, whichemploy the electrical resistance principal of counting and sizing. It isbased on the fact that human cells are poor conductors of electricity,while plasma is a good conductor.

The basic apparatus is shown in my prior PCT patent application, andincludes an aperture tube in which the blood suspension is drawn into anorifice and along an aperture. An electromagnetic field is imposed uponthe aperture, and the blood suspension is drawn through the field. Sincethe liquid of the suspension is essentially homogeneous, and conductive,while the blood cells are resistive, with their resistivity varying withtheir size, the size of the blood cells passing through the aperture maybe calculated by measuring the perturbation of the field as theparticles pass therethrough.

As cells pass through the aperture, the change of voltage that occurs isregistered by the instrument. All instruments known prior to myinventive method (described in my co-pending PCT application) measurethe peak of the impulse produced by the resistance of cell. A specificthreshold is set during calibration which controls the minimum level ofsignal detection. This in turn lowers the presence of the electronic"noise". When the voltage change exceeds the level of the threshold, theinstrument will identify the peak of that impulse. This method iscommonly called the "impedance" or "peak detection method".

To conduct the MRT Test, one needs a very precise measurement of thevolumes of liquids and solids in tested fluid. Common hematologyinstrumentation does not posses high precision for volumetricmeasurement and even though they are accepted in the hematology field,they cannot register very small volumetric changes occurring in cellsduring reactions. For that reason I developed my new (PCT) patentpending method for measuring the volume of solids in a suspension. Likemany hematology instruments, it employs the principal of resistance,illustrated by Ohm's Law:

    V.sub.VOLTAGE =I.sub.CURRENT ×R.sub.RESISTANCE

The new method does not adhere to the standard peak detection. Itcontinuously measures the flow of volume of liquid and solids in thetested liquid.

The actual measurement will appear, if taken graphically, to be the sameas an oscilloscope reading in time and resembles a continuous electricalwave signal (see the actual computer printout identified as FIG. 3).

The series of spikes represent particles causing small disturbances inthe electrical field. The longer and higher the pulse, the greater thevolume of the particle (See printout identified as FIG. 4).

Accordingly, a smaller particle will create a shorter disturbance of asmaller magnitude, and a larger particle will cause a longer disturbanceof a greater magnitude. There is a definite relationship between thelength, height and volume of the tested particle. Since the STS200apparatus measures with a frequency better then 1 MHZ, it is easy toidentify the relationship between the size of the particle and the timeit will need to pass through the orifice. FIG. 5 explains how the MRTmeasurement works and how it differs from the Coulter Method.

The description of the (Prior Art) Coulter Method.

A disturbance caused by particle "P1" produces the spike with the peakhigh's marked "h₁ ". It is measured from the base level up to the peakof the signal. After the particle travels the length of the aperture,the measured signal experiences a "bounce" in which the measured signalgoes below the original baseline, and gradually goes on an upwardgradient towards the original baseline. But a subsequent particle mayoften enter the aperture before the "bounce" is over. For example, inFIG. 5, second particle "P2" starts its disturbance below the staticbase level. The height of h₂ is measured from the baseline and clearlyshows, that the result is not very accurate since the true disturbancecommences below that level. The third particle (P3) is a platelet andits electrical disturbance is entirely below the base level, due to thelarge "bounce" caused by P2, and so is invisible to the instrument.

Disadvantages:

The lower size limit of particles which may be measured is determined bythe static noise threshold established during calibration. The uppersize limit is related to the physical size of the aperture. A majorproblem associated with electric resistance particle counting and sizingbecomes evident when attempts are made to evaluate two dissimilarparticle sizes at the same time using the same aperture, e.g.simultaneous measurement of erythrocytes and platelets. After cells passthrough the orifice, some re-enter the electrical field with the pulseresembling the size of platelets. Threshold and electrical "noises" arealso a substantial problem. A specific constant threshold is set duringthe calibration which controls the minimum level of signal detection.This in turn lowers the presence of the electronic "noise". When thevoltage change exceeds the level of the threshold, the instrument willidentify the peak of that impulse. This is the basis of the peakdetection method.

Description of the MRT (Ribbon) Method.

According to the inventive method, an instrument continuously measuresthe level of the electromagnetic field as the suspension flows throughthe orifice, regardless of the level of the signal. Examples of thesignal measurement are represented by "H" in FIG. 5. Compare thisreading with the prior art method represented by "h". After the particle"P1" passes through the orifice, the signal dips down below thethreshold and the baseline. The Coulter method stops the measurementwhen the signal goes below the threshold level, but the new measurementfollows the signal and measures the time of impulse "P1" which is"V_(s1) ", the time it takes for particle P1 to stop disturbing themeasured signal. The time is measured as the duration of the intervalcommencing when the gradient of the curve begins to indicate thepresence of a particle until the measured signal returns to its originallevel. The presence of the particle is indicated when the gradientincreases for a predetermined period, preferably corresponding to atleast three consecutive measurement clock cycles.

As the particle leaves the orifice, the instrument measures timeidentified as "V_(L2) ". This is the time it takes fluid to pass throughthe orifice. As we approach the point "V_(S2) ", another particle "P2"enters the orifice. The signal is still below the static threshold andthe static baseline, but the STS100 instrument recognizes the conditionand begins to measure the solid particle. This establishes a dynamicbaseline, which is defined as the value of the measured signal when thegradient of the curve begins to increase. The height of the perturbationof the signal is therefore measured as H₂, from the dynamic baseline,rather than from the static baseline as shown by h₂. This moreaccurately reflects the true size of the perturbation of the signal, andtherefore the size of the particle.

The duration of the signal identified as "V_(L2) " is another importantpart of the measurement. If we look at signal "P3". it is evident, thatthe whole impulse is contained below the baseline. The volume of thesolid, identified by time "V_(s3) " arid measured from the dynamicbaseline becomes part of the measurement. The MRT Ribbon Method thuscorrectly measures all particles suspended in the electrolytic solution.There is a definite relationship between the length, height and volumeof the tested particle. Since the STS200 apparatus measures with afrequency greater then 1 MHZ, it is easy to identify the relationshipbetween the size of the particle and the time it will need to passthrough the orifice. Also the flow of fluid is identified.

It has been determined, as well, that the gradient of the curve on theupward slope of the curve when a particle is present also varies withthe size of the particle, larger particles having a steeper slope. Theexact relationship depends upon the configuration of the system, and maybe determined with some minor experimentation depending upon theparameters of the equipment being used. Thus, the gradient may also beused to calculate the size of the particles.

One point should be made about correction of the rough signal shown inFIG. 4. As described in my earlier PCT application, the actual size of aparticle is represented by the "trough" between the peaks of themeasurement curve shown on either the right or left of the figure. Thevalue of the trough is the one selected to represent the correctedheight of the curve.

The flow of fluid is also identified. FIG. 6 graphically represents howthe STS200 identifies the volume of solid and the volume of liquid.

Letter A represents the beginning of the test.

V_(L) =Volume in time in which an instrument measures the liquid V_(S)=Volume in time in which an instrument measure the solid particle. Totalmeasured volume:

    V.sub.T =V.sub.LT +V.sub.ST where

    V.sub.LT =V.sub.L1 + . . . +V.sub.Ln and V.sub.ST =V.sub.S1 + . . . +V.sub.sn

During the measurement, the fluid flows through the orifice. The liquidportion is characterized by the flat impulse line and the solid portionis characterized as the visual disturbance in the flat impulse signal.As the 1 ml (volume of 1 ml is given only as an example) of dilutedblood passes through the orifice, the computer software programquantifies the cumulative volume of liquid and cumulative volume ofsolids in accordance with the rules established, here.

There are at least three different ways of data collection and resultspresentation:

1. Measure 1 ml (or other predetermined volume) of the diluted bloodsample. Calculate the total volume of all solids and subtract them from1 ml. From the difference, the total volume of liquid in the 1 ml ofblood suspension will be given and the volume of liquids in the controland test sample can be compared; or

2. Compare only the total volume of solids in the two samples; or

3. Compare the ratio of solids to liquids (or liquids to solids) in thetwo samples.

Each of these measurements is essentially the same, and any one (ormore) of them may be used at the convenience of the user, as desired.

Step By Step Testing Procedure:

For purposes of visualization I will describe the MRT procedureconducted on the STS200 continuous flow instrument.

After proper test preparation (see section 2), take incubated cuvetteidentified as a "control" and gently mix. Draw 100 μl of diluted bloodand transfer it into empty cuvette. You will have two control cuvettes,one containing 600 μl and another 100 μl of diluted blood. Dispense 10ml of isotonic solution into each cuvette. Additionally add 100 μl oflysing agent to the cuvette containing 600 μl of suspended blood. Placeboth cuvettes on the stage and start the test run. An instrument willmeasure the volume of one ml of the suspended blood in both cuvettes oneafter another and will display detailed information on how manyfemtoliters (fl) of liquid is present in one milliliter of suspendedwhole blood. The next step repeats the preparation process of the samplecuvette. Draw 100 μl of diluted blood from the incubated sample testcuvette. Transfer it into the empty cuvette. Dispense 10 ml of isotonicsolution into each cuvette. Add 100 μl of lysing agent into the cuvettecontaining 600 μl of suspended blood. Place both cuvettes on the stageand run the test. Repeat the cycle for each additional sample tested.Results will be calculated from the information obtained from allsamples, by comparing the total volume of liquid of control sample tothe total volume of liquid of the substance sample. We will obtain tworesults from each substance. One sample will give us information on theactivities of the Red Blood Cells (RBC) and another sample will informus on reactions of all other then RBC blood components in presence oftested substance. It is not mandatory to conduct the MRT Test in thisexact fashion. Per individual need, one can conduct the partial testobtaining results from the first or the second solution only.

4. Results

The computer will establish the volumetric baseline of the plasma(liquid) present in one cubic millimeter of control blood sample. Oncethe baseline is established, the actual volume of plasma present in eachmilliliter of each blood sample will be calculated and compared againstthe actual volume of plasma in the control sample. If liquid volume inthe control sample significantly varies from liquid volume in the testsample, the tested substance is identified as reacted. A significantreaction would be one greater than could be attributed to the knowninstrumentation error plus the standard deviation for similarmeasurements. Any difference of less than that amount would notnecessarily indicate a positive reaction, since it could be attributedto statistical or instrumentation error.

FIG. 7 portrays the measurement of the blood sample distribution of theControl and Test Samples. The differences between the distributionpatterns would be due to the exposure of the Test Sample to the testedsubstance.

The computer program will calculate the variation and save it as theresults data. Interpretation of results will be based on the standarddeviations and other generally accepted laboratory methods of resultsinterpretation.

It will be appreciated by those of ordinary skill in the art that themeasurements of the electromagnetic signal described above may be madeof either the voltage or the current, since it is the resistance withinthe aperture which changes and the imposed field is otherwise constant.

Having described preferred embodiments of the invention with referenceto the accompanying drawings, it is to be understood that the inventionis not limited to those precise embodiments, and that various changesand modifications may be effected therein by one skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

What is claimed is:
 1. An in-vitro method for detecting a reaction inblood caused by substances, comprising the steps of:establishing a firstpotential across a first predetermined spatial volume; passing a firstportion of said blood through said first predetermined spatial volume;substantially continuously measuring said first potential over a firstpredetermined period of time; comparing said measured first potentialwith a first baseline; calculating the total volume of solids in saidfirst portion of said blood as a first function of a total absolutedeviation of said measured first potential from said first baseline;exposing a second portion of said blood to a substance; establishing asecond potential across a second predetermined spatial volume; passingsaid second portion of said blood through said second predeterminedspatial volume; substantially continuously measuring said secondpotential over a second period of time; comparing said measured secondpotential with a second baseline; calculating the total volume of solidsin said second portion of said blood as a second function of a totalabsolute deviation of said measured second potential from said secondbaseline; and comparing said total volume of solids in said secondportion of said blood with said total volume of solids in said firstportion of blood, whereby a positive reaction is established when saidtotal volume of solids in said second portion of blood differs from saidtotal volume of solids in said first portion of blood by more than apredetermined error factor.
 2. The method of claim 1, wherein at leastone of said first and said second substantially continuous measuredpotentials forms a curve, and at least one of said first and secondfunctions is an integral of said curve, so that the total volume ofsolid material measured thereby is a third function of the total areaunder said curve.
 3. The method of claim 1, wherein at least one of saidfirst and second substantially continuous measured potentials forms acurve, and at least one of said first and second functions includesdetermining a gradient of said curve.
 4. The method of claim 3, whereinsaid at least one of said first and second functions further includesidentifying the presence of a solid in the predetermined spatial volumemeasured thereby by comparing changes in said gradient over time.
 5. Themethod of claim 4, wherein the presence of a solid in said predeterminedspatial volume is identified when said comparison of changes in saidgradient shows that said gradient has remained substantially constantfor a third predetermined period of time.
 6. The method of claim 5,wherein said substantially continuous measurement of said potentialcomprises a series of discrete measurements.
 7. The method of claim 6,wherein said series of discrete measurements comprises at least onemillion measurements per second.
 8. The method of claim 7, wherein saidthird period of time is no fewer than three of said discretemeasurements.
 9. The method of claim 5, wherein the presence of a solidto be measured is indicated by a sharp increase in said gradient. 10.The method of claim 9, wherein at least one of said first and secondbaselines is a dynamic baseline located at the value of said measuredpotential at a point on said curve immediately prior to said sharpincrease in said gradient, and said volume of said solid is measuredfrom said dynamic baseline.
 11. The method of claim 10, furthercomprising the step of storing said volume of each said solid.
 12. Themethod of claim 11, further comprising the step of summing the volume ofall measured solids, thereby measuring the total volume of all solids insaid blood.
 13. The method of claim 3, wherein said at least one of saidfirst and second functions includes:storing the value of said curve at apoint when said gradient of said curve increases for more than a fourthpredetermined time interval; and measuring a time duration commencingwhen said gradient increases for more than said fourth predeterminedtime interval until said value of said curve returns to said value. 14.The method of claim 13, wherein said comparison of said potential tosaid baseline yields a height of said curve; andsaid at least one ofsaid first and second functions is derived from at least one of saidtime duration, said gradient of said curve and said height of saidcurve.
 15. The method of claim 1, wherein said first and secondfunctions are identical.
 16. An in-vitro method for detecting a reactionin blood caused by substances, comprising the steps of:establishing afirst potential across a first predetermined spatial volume; passing afirst portion of said blood through said first predetermined spatialvolume; substantially continuously measuring said first potential over afirst predetermined period of time; comparing said measured firstpotential with a first dynamic baseline; calculating the total volume ofsolids in said first portion of said blood as a first function of atotal absolute deviation of said measured first potential from saidfirst dynamic baseline; exposing a second portion of said blood to asubstance; establishing a second potential across a second predeterminedspatial volume; passing said second portion of said blood through saidsecond predetermined spatial volume; substantially continuouslymeasuring said second potential over a second predetermined period oftime; comparing said measured second potential with a second dynamicbaseline; calculating the total volume of solids in said second portionof said blood as a second function of a total absolute deviation of saidmeasured second potential from said second dynamic baseline; andcomparing said total volume of solids in said second portion of saidblood with said total volume of solids in said first portion of blood,whereby a positive reaction is established when said total volume ofsolids in said second portion of blood differs from said total volume ofsolids in said first portion of blood by more than a predetermined errorfactor.
 17. The method of claim 16, wherein said first and secondfunctions are identical.